The Discovery of Novel α2a Adrenergic Receptor Agonists Only Coupling to Gαi/O Proteins by Virtual Screening

Most α2-AR agonists derived from dexmedetomidine have few structural differences between them and have no selectivity for α2A/2B-AR or Gi/Gs, which can lead to side effects in drugs. To obtain novel and potent α2A-AR agonists, we performed virtual screening for human α2A-AR and α2B-AR to find α2A-AR agonists with higher selectivity. Compound P300–2342 and its three analogs significantly decreased the locomotor activity of mice (p < 0.05). Furthermore, P300–2342 and its three analogs inhibited the binding of [3H] Rauwolscine with IC50 values of 7.72 ± 0.76 and 12.23 ± 0.11 μM, respectively, to α2A-AR and α2B-AR. In α2A-AR-HEK293 cells, P300–2342 decreased forskolin-stimulated cAMP production without increasing cAMP production, which indicated that P300–2342 activated α2A-AR with coupling to the Gαi/o pathway but without Gαs coupling. P300–2342 exhibited no agonist but slight antagonist activities in α2B-AR. Similar results were obtained for the analogs of P300–2342. The docking results showed that P300–2342 formed π-hydrogen bonds with Y394, V114 in α2A-AR, and V93 in α2B-AR. Three analogs of P300–2342 formed several π-hydrogen bonds with V114, Y196, F390 in α2A-AR, and V93 in α2B-AR. We believe that these molecules can serve as leads for the further optimization of α2A-AR agonists with potentially few side effects.


Introduction
Alpha2-adrenergic receptors (α 2 -AR) are integral membrane proteins that belong to the superfamily of G-protein-coupled receptors (GPCRs).These receptors regulate effector function via activation of multiple members of Go and Gi G-protein families [1].Three subtypes (α2A/α2-C10, α2B/α2-C2, and α2C/α2-C4) are encoded by distinct genes and widely expressed in the brain.α 2A -AR is most abundant in the rat locus coeruleus, but is also widely localized in the brain stem, cerebral cortex, septum, hypothalamus, hippocampus, and amygdala.α 2B -AR was observed only in the thalamus, while α 2C -AR was mainly distributed in the basal ganglia, olfactory tubercle, hippocampus, and cerebral cortex [2].α 2A -AR is the predominant α 2 -AR subtype in the central nervous system, which prejunctionally inhibits the release of sympathetic, endogenous neurotransmitters such as norepinephrine and epinephrine.They mediate the antihypertensive and the sedative effects of α 2 -AR agonists [3,4].The postjunctional α 2 -AR found in the peripheral tissues has been demonstrated to induce vasoconstriction and increase arterial pressure in various mammalian species [5,6].The α 2B -AR subtype is mainly distributed in the periphery, including lung, kidney, liver, heart, and vascular tissues.It appears to have a dominant role in eliciting the immediate or salt-loading hypertensive response to α 2 -AR agonists [7][8][9].α 2C -AR regulates the release of norepinephrine at low norepinephrine concentrations, in contrast to α 2A -AR regulation at high norepinephrine concentrations [3].The amount of α 2C -AR is lower than that of α 2A -AR in the brain.The autoinhibition mediated by α 2C -AR in α 2C -KO mice could be substituted by α 2A -AR for the elusive character of α 2C -AR [10].
A large amount of structural activity data has emerged in pursuit of the specific agonists and antagonists of α 2 -AR subtypes for potential therapeutics since the discovery of the three α 2 -AR subtypes.Exceptionally, α 2A -AR has a dual pharmacological effect in that it simultaneously couples to Gi and Gs to inhibit or stimulate adenylyl cyclase (AC) activity [11].At low agonist concentrations, α 2A -AR mainly couples to Gi, whereas at high concentrations, Gs coupling dominates.This unusual dual effect has not been well understood for any GPCR.The Gi/o inhibitor pertussis toxin (PTX) pre-treatment inhibited DMED-induced sedation [12], whereas the Gs inhibitor had no effect on the sedation induced by DMED (data not published).The Gs coupling action of these partial agonists of α 2A -AR may not be related to sedation, and the effects remain incompletely understood.α 2A -AR agonists are mainly imidazole ring compounds that can interact with D113 by a salt bridge, and they have no selectivity for α 2A/2B -AR or Gi/Gs [13].To enhance the therapeutic potency of α 2 agonists as antihypertensive and sedative drugs, compounds should bind to α 2B -AR with lower affinity or have no coupling to Gs proteins at high concentrations.
Computer-aided drug design has been widely used to discover novel and potent lead compounds.This study combined crystal structures for human α 2A -AR [14] and α 2B -AR [15] with virtual screening to obtain highly selective α 2A -AR agonists as potential sedatives.The ChemDiv compound library (1,535,478 compounds) was screened, and 1023 compounds were found that could bind to α 2A -AR with high affinity but low affinity to α 2B -AR.We selected 105 compounds with good druggability according to the virtual screening; these molecules were studied in the loss of righting reflex (LORR) of C57/B6 mice.Compound P300-2342 and three analogs had a sedative effect after α 2A -AR activation, as did the Gi/o-coupled pathway with the inhibition of AC-cyclic adenosine monophosphate (cAMP) accumulation.These compounds could serve as leads for sedation by α 2A -AR activation with potentially few side effects.

Analysis of the Molecular Docking Results
All 50,679,311 molecular conformations generated by the Chemdiv compounds library (Version 2019) were docked to α 2A -AR, and the top 100,000 compounds with high affinity were then docked to α 2B -AR.The molecular weight distribution of these compounds ranged from 216 Dal to 767 Dal.The binding energy [ChemGauss Score (CGS)] of these compounds to α 2A -AR ranged from −22 to −16 kcal/mol, and ranged from −19 to −8 kcal/mol for α 2B -AR.A correlation analysis showed that the binding affinity of these compounds for the α 2A -AR receptor did not correlate to that for α 2B -AR [Figure S1], which made it possible to select the compounds that may bind to α 2A -AR with high affinity but not to α 2B -AR or with low binding affinity.The binding energies of the selected compounds to α 2A -AR or α 2B -AR ranged from −22 to −16 kcal/mol or −14 to −8 kcal/mol, respectively.In addition, compound C593-0297 was exceptionally selected with the binding energy −14.85 kcal/mol to α 2B -AR [red dot in Figure S1].Finally, 1023 compounds from the Chemdiv database were screened for druggability filtrates.

Properties of the Selected Compounds
StarDrop software (6.5.0) was used to analyze the properties of 1023 compounds including water solubility (logS), the lipid-water distribution coefficient (logP), molecular weight, molecular flexibility, the hydrogen bond, the topological polar surface access area, the cytochrome P450 2C9 (CYP2C9) enzyme degradation level, the human ether-à-go-gorelated gene (hERG) value, oral availability, drug interaction risk (2D6), and other indicators.The summarized score (SS) was calculated based on these properties.The SS values of these compounds ranged from the largest, 0.4379, to the smallest, 0.0023.According to the SS values, the top 500 compounds with the highest SS scores were selected for the following analysis.
A cluster analysis was performed by a maximum common substructure algorithm to obtain structural diversity of candidate compounds; the similarity threshold was set to 0.5, which divided the 500 compounds into 67 clusters.The correlation plots between the SS scores and other properties such as binding affinity, logS, and logP were analyzed the SS values, the top 500 compounds with the highest SS scores were selected for the following analysis.
A cluster analysis was performed by a maximum common substructure algorithm to obtain structural diversity of candidate compounds; the similarity threshold was set to 0.5, which divided the 500 compounds into 67 clusters.The correlation plots between the SS scores and other properties such as binding affinity, logS, and logP were analyzed
DMED addition; data are shown as the mean ± S.E.M value of three independent experiments performed in triplicate.

Discussion
The selective α2-AR subtype antagonist and α2-AR subtype knockout mice demonstrated α2A-AR-subtype-mediated sedation induced by α2-AR agonists [16].Presynaptic α2A-AR activation of Gi/o can inhibit the activity of adenylate cyclase with the attenuation of cAMP production, activation of K + conduction, acceleration of Na + /H + exchange, and inhibition of Ca 2+ channels [17].As the classic GPCR, α2A-AR has seven transmembrane segments, three extracellular loops, and three intracellular loops.Each transmembrane segment consists of 20 to 26 amino acids forming an α helix, in which the hydrophobic amino acids form a "pocket", which determines the specificity of binding to the ligand.The second and third intracellular loops are related to the G protein coupling of the receptor.The N-terminal part of the third intracellular loop and some amino acid residues of the second intracellular loop determine the specificity of the interaction between the receptor and the G protein.The C-terminal part of the third intracellular loop is also involved in the interaction between the receptor and the G protein, but it is mainly related to the coupling efficiency of the receptor and the G protein [11,18].
The Chemdiv database (Version 2019), containing 1,535,478 small molecule compounds with diverse backbones, was used for virtual screening.To ensure the global

Discussion
The selective α 2 -AR subtype antagonist and α 2 -AR subtype knockout mice demonstrated α 2A -AR-subtype-mediated sedation induced by α 2 -AR agonists [16].Presynaptic α 2A -AR activation of Gi/o can inhibit the activity of adenylate cyclase with the attenuation of cAMP production, activation of K + conduction, acceleration of Na + /H + exchange, and inhibition of Ca 2+ channels [17].As the classic GPCR, α 2A -AR has seven transmembrane segments, three extracellular loops, and three intracellular loops.Each transmembrane segment consists of 20 to 26 amino acids forming an α helix, in which the hydrophobic amino acids form a "pocket", which determines the specificity of binding to the ligand.The second and third intracellular loops are related to the G protein coupling of the receptor.The N-terminal part of the third intracellular loop and some amino acid residues of the second intracellular loop determine the specificity of the interaction between the receptor and the G protein.The C-terminal part of the third intracellular loop is also involved in the interaction between the receptor and the G protein, but it is mainly related to the coupling efficiency of the receptor and the G protein [11,18].
The Chemdiv database (Version 2019), containing 1,535,478 small molecule compounds with diverse backbones, was used for virtual screening.To ensure the global conformation of small molecules in the virtual screening process, the plug-in omega in OpenEye software was used to generate small molecule conformations.On average, each small molecule generated about 50 conformations.We converted the Chemdiv molecules into 50,679,311 molecular conformations and docked the compounds with α 2A -AR crystal structures, and the top 100,000 were then docked to α 2B -AR crystal structures.There was no obvious correlation in the compound affinities to α 2A -AR and α 2B -AR [Figure S1].The Chemdiv database binding the α 2A -AR receptor with high affinity but with low binding affinity to α 2B -AR allowed us to select 1023 compounds for property analysis.The 500 compounds with the highest SS scores (logS, logP, molecular weight, molecular flexibility, hydrogen bond, topological polar surface area (TPSA), CYP2C9 degradation, hERG inhibition, HIA, 2D6, etc.) were selected for structural diversity analysis.The 140 compounds with the best SS values and diverse structures were further classified into 21 categories with an enhanced similarity threshold (0.7) [Figure 1].
In our study, approximately three quarters of the 105 compounds with the best SS values had no sedation or anti-sedative effects, one quarter of the compounds had sedative effects, and a few compounds had anti-sedative effects [Figure 2A, Table S1].Most of the small molecular compounds interacted with D113 in the third transmembrane helix and E189 in the second extracellular loop.At the same time, N93 and V114 were also involved in the binding of most compounds.In addition, 17 amino acid sites, including W99, C106, Y109, C117, T118, S165, C188, I190, D192, Y196, S200, F390, F391, Y394, K409, F412, and Y416, were involved in the interaction of the compounds and α 2A -AR in the present study.D113 in the third transmembrane domain (TM3) is the conservative ligand-contacting residue in the aminergic receptor including α 1 , α 2 , and the β adrenergic receptor [19].D113N inhibits the binding of [ 3 H] yohimbine to α 2 -adrenergic receptors with a small decrease in cAMP accumulation stimulated by forskolin and the potentiation of forskolinstimulated cAMP accumulation [13].S200 in the fifth transmembrane domain (TM5) of hα 2 -AR involved in the ligand binding pocket plays an important role in epinephrine and UK-14304 binding and response [13,20].Qu et al. found twelve residues (D113, V114, S200, C201, S204, W387, F390, F391, Y394, F408, F412, and Y416) involved in the binding pocket of α 2A -AR by a co-crystallized partial agonist and antagonist.These four residues, D113, F390, F412, and Y416, are involved in the formation of an aromatic cage in both α 2A -AR and α 2B -AR [14].In our study, Y394 in α 2A -AR formed a π-hydrogen bond with an imidazoline ring in compound P300-2342.Y394 formed hydrogen bonds with the α 2A -AR agonists epinephrine, norepinephrine, and UK14,304 [14].V114 in the third transmembrane helix of α 2A -AR also formed a π-hydrogen bond with a phenyl ring in P300-2342.P300-2342 also formed a π-hydrogen bond with Val93 in α 2B -AR receptors (Figure 4(A3)).
Similar to previous reports, α 2A -AR wild type (WT) simultaneously coupled to both the Gi and Gs proteins [21] when stimulated by the agonists UK14,304 and DMED (Figure 5A).However, α 2A -AR WT activated by P300-2342 only coupled to Gi proteins (Figures 5A and 6A).In contrast to α 2A -AR and Y394N in previous reports [14], the mutant α 2A -AR Y394A lost the coupling to Gi proteins and only coupled to Gs proteins with DMED treatment [Figure 5D].In the epinephrine-bound β2-AR, epinephrine formed hydrogen bonds with S204 and N293 (6.55), which play an important role in the Gs activation of β2-AR.N293 is relative to Y394 (6.55) in α 2A -AR [22].The polar amino acids Y394 or N394 in α 2A -AR are important to Gi protein signals under agonist addition.In contrast, the nonpolar amino acid A394 mutant α 2A -AR only activated Gs protein signals in our study.
GPCR signaling is well-demonstrated according to the progress in GPCR structural biology.The GPCR cytoplasmic end of the seven transmembrane helix domain is closed in the inactive states [23,24].The sixth transmembrane helix plays an important role in GPCR-Gi activation [25][26][27].In the present study, α 2A -AR Y394A in the mutant sixth transmembrane helix lost Gi protein activation upon agonist addition, in accordance with the previous view.Except for the amino in transmembrane helix 6, the three serines (S200, S201, and S204) in transmembrane helix 5 and D130 in transmembrane helix 3 are involved in the agonist interaction with β2-AR [22,28,29].These amino acids are also involved in α 2 -AR interactions with catecholamines [30], which might play an important role in Gs protein coupling.P300-2342 formed a π-hydrogen bond with Y394 in the sixth transmembrane helix and V114 in the third transmembrane helix in the binding pockets of α 2A -AR.The interaction between P300-2342 and α 2A -AR could not make the receptor couple to the Gs protein.Consistent with the α 2A -ARY394A mutant, α 2A -AR V114A only coupled to Gs proteins after DMED addition [Figure 6C].P300-2342 lost its coupling to Gi proteins in α 2A -AR, Y394A, α 2A -AR, and the V114A mutant, which indicated that V114 in the third and Y394 in the sixth transmembrane helixes play important roles in Gi protein coupling (Figure 6C,D).Although P300-2342 formed a π-hydrogen bond with V93 in α 2B -AR and inhibited the binding of [ 3 H] Rauwolscine to α 2B -AR, P300-2342 had no activation in α 2B -AR coupling to Gs proteins.V93 is a conserved site in α 1 , α 2 , and β-AR [15].Yuan et al. found V93 in the third transmembrane helix involved in the binding pocket of DMED in α 2B -AR, but V93 had no interaction with DMED [15].V93 might not to be the key site for the agonist activation.The P300-2342 interacting sites Y394, V114 in α 2A -AR, and V93 in α 2B -AR contributed to the uncoupling to Gs proteins, which prevented the unwanted Gs coupling activity.The P300-2342 analogs T226-1970, P300-2344, and P300-1500 also formed π-hydrogen bonds or hydrogen bonds with α 2A -AR and α 2B -AR, and then inhibited the binding of [ 3 H] Rauwolscine to α 2A -AR and α 2B -AR.α 2A -AR only coupled to Gi proteins following the activation of three analogs of P300-2342.T226-1970, P300-2344, and P300-1500 also had no activation to α 2B -AR (Figure 6B).The structure of P300-2342 and the three analogs is more complex than that of the α 2A -AR agonists like epinephrine, dexmedetomidine, and clonidine [Figure 2].These compounds were also larger than the α 2A -AR full agonist UK14304.P300-2342 and its analogs had several benzene moieties with a long polar residue.These large molecular compounds were hydrophobic and obstructed the interaction with the residues in α 2A -AR.Therefore, α 2A -AR could not couple to Gs proteins after being activated by P300-2342 and its analogs.P300-2342 and its analogs could serve as lead compounds with sedative effects without the vascular side effects.We speculate that the future development of compounds capable of selectively activating α 2A -AR while blocking α 2B -AR may further improve our capability to treat hypertension, ischemic heart disease, and heart failure.

Conclusions
Most α 2 -AR agonists are imidazole ring compounds that have no selectivity for α 2A/2B -AR or Gi/Gs, potentially resulting in drug side effects.In this study, we performed a structure-based virtual screening strategy to identify the selective α 2A -AR agonists.We used virtual screening and druggability filtering for the ChemDiv library.We found that 1023 compounds may bind to α 2A -AR with high affinity but with low affinity to α 2B -AR.Among them, 105 compounds with good druggability according to virtual screening were purchased and studied in the LORR of C57/B6 mice.Ligand binding assays showed that compound P300-2342 selectively activated α 2A -AR, and the cAMP assay indicated that P300-2342 could only couple to the Gi/o protein signaling pathway for sedation.Compound P300-2342 may serve as a lead compound for sedatives by activation of α 2A -AR with potentially few side effects.Our research can provide a basis for developing novel selective agonists.

Protein Preparation for Humanα 2 -AR Proteins and Binding Pocket Identification
The 3D structure of α 2A -AR in complex with a partial agonist E39 (pdbid:6kuy) is shown in Figure S3A, where the ligand binding area is in the extracellular domain.The ligand binding area contained four adjacent hydrophobic pockets [Figure S4A].Similarly, the 3D structure of α 2B -AR in complex with agonist DMED (pdbid:6k42) is shown in Figure S3B, with seven transmembrane regions and an extracellular domain, which was highly similar to the structure of α 2A -AR subtype (all atoms RMSD = 4.27 Å).The ligandbinding site was located in the extracellular domain, which is consistent with the ligandbinding region of the α 2A -AR subtype.Two adjacent molecular binding pockets were selected at the ligand site in crystal structures by the MOE package [Figure S4B].In order to identify the compounds which could specifically bind to the α 2A -AR subtype but not α 2B -AR, the relevant pockets from both subtypes were chosen to perform the structure-based virtual screening.

Virtual Screening
The crystal structure of α 2A -AR and α 2B -AR were protonated and optimized by MOE QuickPrep (2019.0102).The optimized structures were then converted to the formatted receptor files that defined all the above-mentioned residues as the docking area by using the appropriate (apopdb2receptor) module of OpenEye software (Release 3.2.0.2).The docking areas of α 2A -AR and α 2B -AR were a rectangle with a box size of 27.67 Å × 19.33Å × 22.33 Å and 28.00 Å × 23.67 Å × 23.67 Å, respectively.
The Chemdiv compound library was selected for virtual screening, which contained 1,535,478 compounds.FRED performs rigid docking, so OMEGA2 was used with all default parameters to search for as many conformations as possible for each compound.OEDocking (Release 3.0) was used to dock all conformations of compounds to α 2A -AR, with the parameters -save_component_scores: true; -hitlist_size: 100,000; -docked_molecule_file: sdf.The top 100,000 compounds for α 2A -AR were docked to the binding pocket of α 2B -AR, and multiple conformations were also generated by OMEGA.StarDrop (Release 6.6.4) was used to calculate the structural diversity based on a maximum common substructure algorithm to cluster compounds containing a significant common substructure.The molecular properties such as logS, HIA category, logP, hERG, 2D6, 2C9 affinity, P-gp category, PPB90, and BBB permeability were used as a scoring function to evaluate the druggability of each molecule.

Materials
C57BL/6 mice (20-22 g) were purchased from the SPF biotechnology co., LTD (Beijing, China).All the animals were housed in an alternating 12 h light/12 h dark cycle room, which was maintained in a climate-controlled environment (25 ± 1 • C).Animals had free access to water and food.All the experimental procedures were reviewed and approved by the ethics committee and institutional animal care and use committee of the Beijing Institute of Pharmacology and Toxicology, Beijing, China (IACUC of AMMS-06-2017-001).Animals were assigned to groups randomly before testing.Testing occurred within the same approximate time of day between experiments.The experimenter was blinded to treatment throughout the course of behavioral testing.
Drugs were purchased from various vendors.Dexmedetomidine was purchased from (Dexinjia Biotechnology, Jinan, China).BRL44408 was purchased from (Sigma, Louis, MO, USA).Forskolin was purchased from (Sigma, Louis, MO, USA).UK 14,304 was purchased from (Tocris, Bristol, UK).[ 3 H] Rauwolscine was purchased from (PerkinElmer, Waltham, MA, USA).The selected compounds were purchased from (Topscience, Shanghai, China).Dexmedetomidine and BRL44408 were dissolved in sterile physiological saline (0.9% NaCl) to the final concentration used in this study.The selected compounds were dissolved in 5% DMSO.

LORR and Locomotor Activity
The LORR in C57/B6 mice were used to study the effects of screening compounds.Mice were pre-treated with compounds (100 nmol/mouse, i.c.v.) 15 min prior to DMED administration (432 nmol/kg, i.p.).The righting reflex was deemed to be "lost" if the mouse failed to right itself within 60 s of being placed on its back, as described in our previous study [31].The effects of the compounds on the spontaneous locomotor activity of mice were detected using a LabState video tracking system (Anilab Scientific Instruments Co., Ltd., Ningbo, China).The mice were placed in acrylic boxes (40 cm × 40 cm × 35 cm) for 30-60 min before compound treatment on day 1.On day 2, compounds (10 or 100 nmol) were injected into the cerebral ventricles (i.c.v.) of the mice 30 min before being placed in acrylic boxes.The locomotor activity of each mouse was defined by the horizontal distance it travelled (in centimeters) over 30-60 min [32].

cAMP Assay
According to the GloSensor cAMP biosensor (Promega, Shanghai, China) manufacturer protocols, HEK293 cells were co-transfected with the pGloSensor-22F cAMP plasmid (Promega, E1171) and pCMV6 Entry-Flag-α 2A -AR.After 24 h, the cells were seeded in white 96-well plates.The next day, the cell media were replaced by 90 µL of fresh DMEM with 2% v/v GloSensor cAMP reagent (Promega, E1290) and incubated for 60 min at 37 • C. Before drug stimulation, an initial measurement of the baseline signal was detected.Then cells were stimulated with 10 −12 , M-10 −5 , M, P300-2342, T226-1970, P300-2344, P300-1500, or DMED for 10 min and then were stimulated with forskolin (10 µM) for 15 min with or without α 2A -AR antagonist BRL44408 (10 µM) pretreatment.PTX 500 ng/mL selectively ablated receptor Gi coupling [32].PTX 500 ng/mL pretreatment for 24 h was used to assay the chemical effects on Gi coupling following α 2A -AR activation.Two mutations, V114A and Y394A were introduced into the α 2A -AR genes, respectively, using standard QuickChange PCR.The cAMP accumulations induced by DMED or P300-2342 were also studied in HEK293 cells co-transfected with the pGloSensor-22F cAMP plasmid and pCMV6 Entry-Flag-α 2A V114A-AR or α 2A Y394A-AR with forskolin (10 µM) stimulation.HEK293 cells co-transfected with the pGloSensor-22F cAMP plasmid and pCMV6 Entry-Flag-α 2B -AR were stimulated with P300-2342, T226-1970, P300-2344, P300-1500, or DMED to detect the cAMP accumulation.In each experiment, the cAMP assay used pcDNA3.1 myc/hisB transfected cells as a negative control and cells stimulated with 10 µM of forskolin as a positive control.The signal was read using a Victor 2D Instrument (PerkinElmer) at 675 nm.The production of cAMP% was calculated (the signal value after chemicals addition-baseline signal value)/the baseline signal value *100 as described in our previous study [33].
[Figure S2].By analyzing the structural diversity, binding affinity, and druggability scores, 140 compounds with the best SS values and diverse structures were kept [red in Figure S2A], which were further classified into 21 clusters with an enhanced similarity threshold (0.7) [Figure 1].Most of the categories contained only two-three compounds, but cluster 2, 4, 11, and 12 contained more compounds.The compounds from cluster 4 and 16 showed high binding affinity to α 2A -AR [Figure 1A].Compared with other clusters, compounds 4 and 10 also bound to α 2B -AR [Figure 1B].Compounds from cluster 7, 9 and 14 may have had lower water solubility [Figure 1C].Int.J. Mol.Sci.2024, 25, x FOR PEER REVIEW 3 of 16

Figure 1 .
Figure 1.The properties of 140 compounds in 21 categories.The compounds' affinity to the α2A adrenergic receptor (A), α2B adrenergic receptor (B), and LogS (C) of compounds in 21 categories.

Figure 1 .
Figure 1.The properties of 140 compounds in 21 categories.The compounds' affinity to the α2A adrenergic receptor (A), α2B adrenergic receptor (B), and LogS (C) of compounds in 21 categories.

Figure 2 .
Figure 2. The effects of compounds on the LORR and locomotor activity in mice.(A) A total of 105 compounds (100 nmol/mouse, i.c.v.) 15 min prior to DMED administration (432 nmol/kg, i.p.).Twenty-eight compounds increased (active) and four compounds decreased (negative) the percent of LORR induced by DMED (432 nmol/kg, i.p.).Seventy-three compounds displayed no effects (inactive).(B) The effect of the active compounds (100 nmol/mouse, i.c.v.) on the locomotor activity of mice.P300-2342 was red.(C) The effects of atipamezole (ATI,0.2mg/kg, i.p.) on the locomotor activity inhibited by four compounds and DMED (10 nmol/mouse, i.c.v.).The red column showed the antagonism of ATI on P300-2342.(D) The locomotor activity of mice induced by 13 analogs of P300-2342.n = 8, mean ± SEM, * p< 0.05, ** p < 0.01 compared with the solvent.The analysis was performed using a one-way ANOVA followed by Dunnett's test (B,D) and a two-way ANOVA followed by Sidak's test (C).